THE ROLE OF CORE-BINDING FACTOR SUBUNIT BETA (CBFβ) AND THE RUNT-RELATED TRANSCRIPTION FACTOR (RUNX) FAMILY IN MALE GERMLINE DEVELOPMENT

Mustika Rahmawati

doi:10.7273/000007450

The precise regulation of spermatogenesis, involving a tightly controlled balance between spermatogonial proliferation and differentiation, is essential to support continuous sperm production and male fertility. Disruptions to this balance can impair germ cell maturation and compromise fertility. Using conditional knockout mouse models, our study revealed the previously unexplored critical role of the Core-Binding Factor Subunit Beta (CBFβ) as a transcriptional cofactor in maintaining male fertility through the regulation of germ cell proliferation and differentiation during prepubertal development. Our study also unravels the functional role of CBFβ in maintaining a delicate balance between proliferation and differentiation that is established early in development and necessary to complete terminal differentiation from the undifferentiated spermatogonia population. Furthermore, using spatial transcriptomic, we identified critical cell cycle regulatory genes as targets of CBFβ in the germline, suggesting its involvement in the overall cell cycle control during spermatogonial proliferation and meiotic progression. To further investigate the intricate balance that CBFβ has to maintain between proliferation and differentiation, we examined the functional partners of CBFβ. CBFβ is an established cofactor for the Runt-related Transcription Factor (RUNX) family in many developmental processes. Investigations into the interaction of CBFβ with the RUNX proteins identified RUNX1 and RUNX3 as binding partners of CBFβ in the male germline. Protein expression profiling also revealed distinct expression patterns of RUNX1 and RUNX3, particularly regarding their temporal dynamics during specific cell cycle phases in the undifferentiated spermatogonia. Notably, RUNX1 expression is elevated in proliferative germ cells, reaching its peak during the late stages of the cell cycle (G2/M phases). Using CUT&RUN, we identified shared targets among CBFβ, RUNX1, and RUNX3 through Runt motif binding domain. In addition, we detected enrichments of other motifs belonging to transcription factors involved in proliferation and differentiation in targets shared among CBFβ, RUNX1, and RUNX3. Moreover, we uncovered distinct yet complementary targets involved in proliferation and differentiation processes by CBFβ-RUNX1and CBFβ-RUNX3, respectively. Interestingly, we also identified RUNX-independent targets of CBFβ, indicating a broader cell cycle regulatory role of CBFβ involving other unidentified partners. Here, our study suggests that CBFβ, RUNX1, and RUNX3 create an interconnected network of transcriptional regulation in the undifferentiated spermatogonia to balance proliferation and differentiation. Together, this study advances our understanding of the molecular mechanisms underlying postnatal male germline regulation by emphasizing the functional role of CBFβ and its interactions with RUNX and other co-regulatory proteins. Collectively, this study further illuminates the complex regulatory networks in the prepubertal germline development essential to support lifelong spermatogenesis and male fertility.

THE ROLE OF CORE-BINDING FACTOR SUBUNIT BETA (CBFβ) AND THE RUNT-RELATED TRANSCRIPTION FACTOR (RUNX) FAMILY IN MALE GERMLINE DEVELOPMENT

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